Journal: bioRxiv

Article Title: Microvasculopathy And Soft Tissue Calcification In Mice Are Governed by Fetuin-A, Pyrophosphate And Magnesium

doi: 10.1101/577239

Figure Lengend Snippet: a, b, Wildtype (wt) and c, d, fetuin-A-deficient ( Ahsg−/− ) mice maintained against the genetic background DBA/2 (D2) or C57BL/6 (B6) were analyzed by computed tomography, and calcified lesions were segmented (red color) and e, quantified. Arrows point to small calcified lesions in a, the interscapular brown fat tissue of D2,wt and d, B6, Ahsg−/− mice. c, Arrows depict from left to right, massive calcified lesions in the left kidney, the heart, the interscapular brown adipose tissue BAT, and the axillary skin. f-i, Serum chemistry of calcification related electrolytes demonstrated f, normal serum total calcium, g, normal serum phosphate Pi, h, hypomagnesemia in D2,wt mice compared to B6,wt mice, and failure to induce serum magnesium in D2, Ahsg−/− compared to B6, Ahsg−/− , resulting in functional hypomagnesemia, i, plasma pyrophosphate, PPi was highly variable, yet slightly elevated in B6 mice compared to D2 mice and j, plasma FGF23 was significantly elevated in D2 compared to B6 mice, regardless of fetuin-A genotype. One-way ANOVA with Tukey’s multiple comparison test for statistical significance, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Serum FGF-23 was measured with ELISA (Mouse Fibroblast growth factor 23 ELISA Kit, Cusabio, CSB-EL008629MO) according to manufacturers instructions, with samples diluted 1:2 in sample diluent.

Techniques: Computed Tomography, Functional Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway

doi: 10.3389/fcvm.2023.990422

Figure Lengend Snippet: Fibroblast growth factor 23 increases mRNA levels of markers of hypertrophy and fibrosis. Rat cardiac myoblast cells (H9c2) were cultured with 0, 50, or 100 ng/mL fibroblast growth factor 3 (FGF23) for 24 h, and mRNA expression levels were analyzed by real-time PCR. (A) Atrial natriuretic factor (ANF) ( n = 6). ** P < 0.05 versus 0 ng/mL. (B) Brain natriuretic peptide (BNP) ( n = 6). * P < 0.01 versus 0 ng/mL. (C) β-myosin heavy chain ( beta MHC ) ( n = 6). ** P < 0.05 versus 0 ng/mL. (D) Alpha smooth muscle actin ( alpha SMA ) ( n = 6). * P < 0.01 versus 0 ng/mL. (E) Collagen I ( n = 6). * P < 0.01 versus 0 ng/mL. (F) FGF23 ( n = 6). * P < 0.01 versus 0 ng/mL. Data were analyzed by one-way analysis of variance.

Article Snippet: The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA).

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cardiovascular Medicine

Article Title: Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway

doi: 10.3389/fcvm.2023.990422

Figure Lengend Snippet: Indoxyl sulfate increases mRNA levels of markers of hypertrophy and fibrosis. H9c2 cells were cultured with 0, 0.25, or 1.0 mM indoxyl sulfate (IS) for 24 h, and mRNA expression levels were analyzed by real-time PCR. (A) Atrial natriuretic factor ( ANF ) ( n = 6). * P < 0.01 versus 0 mM. (B) Brain natriuretic peptide (BNP) ( n = 6). * P < 0.01 versus 0 mM. (C) β-myosin heavy chain ( beta MHC ) ( n = 6). ** P < 0.05 versus 0 mM. (D) Alpha smooth muscle actin ( alpha SMA ) ( n = 6). ** P < 0.05 versus 0 mM. (E) Collagen I ( n = 6). ** P < 0.05 versus 0 mM. (F) FGF23 ( n = 6). ** P < 0.05 versus 0 mM. Data were analyzed by one-way analysis of variance.

Article Snippet: The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA).

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cardiovascular Medicine

Article Title: Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway

doi: 10.3389/fcvm.2023.990422

Figure Lengend Snippet: Indoxyl sulfate increases fibroblast growth factor 23 protein expression and fibroblast growth factor receptor 4 phosphorylation. (A,B) Western blotting of fibroblast growth factor 3 (FGF23) protein expression in H9c2 cells. H9c2 cells were cultured with 0, 0.25, or 1.0 mM indoxyl sulfate (IS) for 72 h. Alpha, beta-tubulin protein expression was examined as an internal control ( n = 5). * P < 0.05 versus IS 0 mM. (C,D) Western blotting of fibroblast growth factor receptor 4 (FGFR4) protein expression and FGFR4 phosphorylation in H9c2 cells. H9c2 cells were cultured with 0, 0.25, or 1.0 mM IS for 72 h. Alpha, beta-tubulin protein expression was examined as an internal control ( n = 5). * P < 0.05 versus IS 0 mM. (E–G) Western blotting of furin protein in H9c2 cells. H9c2 cells were cultured with 0 or 1.0 mM IS for 72 h. The images are from different parts of the same gel. Beta actin protein expression was examined as an internal control ( n = 6). Furin stained as two bands, furin precursor (96 KDa) and mature (90 KDa) forms. * P < 0.05 versus IS 0 mM. Data were analyzed by one-way analysis of variance.

Article Snippet: The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Cell Culture, Control, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway

doi: 10.3389/fcvm.2023.990422

Figure Lengend Snippet: Body weight, blood pressure, and laboratory data from animal experiments.

Article Snippet: The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA).

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway

doi: 10.3389/fcvm.2023.990422

Figure Lengend Snippet: (A,B) IS increases intact FGF23 expression in the heart. The expression of intact FGF23 in the heart was significantly increased after IS treatment. This effect was abrogated by FGFR4 inhibition as shown by Western blotting ( n = 3–6). Data were analyzed by one-way analysis of variance. * P < 0.05.

Article Snippet: The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Inhibition, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway

doi: 10.3389/fcvm.2023.990422

Figure Lengend Snippet: IS induces FGF23 via AhR in vitro . H9c2 cells were cultured with 0 or 1 mM IS for 6 h (for mRNA) or 48 h (for protein) after pretreatment with AhR siRNA or control siRNA (ctrl). (A) FGF23 mRNA levels were significantly increased by IS and downregulated with AhR siRNA ( n = 11). * P < 0.01, ** P < 0.05. (B,C) Western blotting of FGF23 protein expression in H9c2 cells. Alpha, beta-tubulin protein expression was examined as an internal control ( n = 6). * P < 0.01, ** P < 0.05. (D,E) Western blotting of HIF1α protein expression in H9c2 cells. The images are different parts of the same gel. Alpha, beta-tubulin protein expression was used as an internal control ( n = 6). * P < 0.01, ** P < 0.05. (F) H9c2 cells were cultured with 0, 50, or 100 ng/mL FGF23 for 24 h, and mRNA expression levels were analyzed by real-time PCR. Levels of polypeptide N-acetylgalactosaminyltransferase 3 ( GALNT3 ) mRNA ( n = 6). * P < 0.01, ** P < 0.05. (G) H9c2 cells were cultured with 0, 0.25, or 1.0 mM IS for 24 h, and mRNA expression levels were analyzed by real-time PCR. Levels of GALNT3 mRNA ( n = 6). * P < 0.01, ** P < 0.05. (H) Levels of GALNT3 mRNA ( n = 9). * P < 0.01, ** P < 0.05. (I,J) Western blotting of GALNT3 protein expression in H9c2 cells. Alpha, beta-tubulin protein expression was used as an internal control ( n = 6). * P < 0.01, ** P < 0.05. Data were analyzed by one-way analysis of variance.

Article Snippet: The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA).

Techniques: In Vitro, Cell Culture, Control, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cardiovascular Medicine

Article Title: Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway

doi: 10.3389/fcvm.2023.990422

Figure Lengend Snippet: IS increases hypertrophic markers via FGF23 in vitro . H9c2 cells were cultured with 0 or 1 mM IS for 24 h after pretreatment with FGF23 siRNA or control siRNA (ctrl), and mRNA expression levels were analyzed by real-time PCR. (A) ANF , (B) BNP , (C) beta MHC , (D) alpha SMA , (E) collagen I, and (F) FGF23 mRNA levels were significantly increased by IS and downregulated with FGF23 siRNA ( n = 6). Data were analyzed by one-way analysis of variance. * P < 0.01, ** P < 0.05.

Article Snippet: The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA).

Techniques: In Vitro, Cell Culture, Control, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cardiovascular Medicine

Article Title: Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway

doi: 10.3389/fcvm.2023.990422

Figure Lengend Snippet: IS increases FGFR4 via FGF23 in vitro . H9c2 cells were cultured with 0 or 1 mM IS for 48 h after pretreatment with FGF23 siRNA or control siRNA (ctrl). (A,B) Western blotting of FGF23 protein expression in H9c2 cells. (C,D) Western blotting of FGFR4 protein expression and FGFR4 phosphorylation in H9c2 cells. Alpha, beta-tubulin protein expression was examined as an internal control ( n = 6). Data were analyzed by one-way analysis of variance. * P < 0.01, ** P < 0.05.

Article Snippet: The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA).

Techniques: In Vitro, Cell Culture, Control, Western Blot, Expressing, Phospho-proteomics

Journal: Frontiers in Cardiovascular Medicine

Article Title: Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway

doi: 10.3389/fcvm.2023.990422

Figure Lengend Snippet: Potential mechanism by which IS may induce LVH via the FGF23-FGFR4 pathway. IS upregulates FGF23 mRNA via AhR and HIF1α. IS also increases GALNT3 gene expression, preventing furin-mediated degradation of intact FGF23. Increased FGF23 induces myocardial hypertrophy by FGFR4-dependent activation.

Article Snippet: The following antibodies were used as primary antibodies: monoclonal anti-rat FGF23 antibody (1:500, MAB2629; R&D Systems); polyclonal anti-goat FGF23 antibody (1:1000, ab123502; Abcam, Cambridge, UK); polyclonal anti-rabbit FGFR4 antibody (1:1000, ab119378; Abcam); polyclonal anti-rabbit FGFR4 (phospho Y642; pFGFR4) antibody (1:1000, ab192589; Abcam); polyclonal anti-rabbit furin antibody (1:1000, PA1-062; Thermo Fisher Scientific); polyclonal anti-rabbit hypoxia-inducible factor 1 alpha (HIF1α) antibody (1:1000, NB100-134; Novus Biologicals, Centennial, CO); polyclonal anti-rabbit polypeptide GALNT3 antibody (1:1000, SAB2106736; Sigma-Aldrich); and anti-rabbit α/β tubulin (1:1000, CST#2148; Cell Signaling Technology, Danvers, MA).

Techniques: Gene Expression, Activation Assay

Journal: PLoS ONE

Article Title: High phosphate intake induces bone loss in nephrectomized thalassemic mice

doi: 10.1371/journal.pone.0268732

Figure Lengend Snippet: (A) Serum erythropoietin from sham WT (n = 7), nephrectomized WT (n = 5), nephrectomized WT+PBS (n = 8), sham BKO (n = 5), nephrectomized BKO (n = 4), and nephrectomized BKO+PBS mice (n = 3). (B) Serum FGF23 from sham WT (n = 7), nephrectomized WT (n = 7), nephrectomized WT+PBS (n = 8), sham BKO (n = 5), nephrectomized BKO (n = 5), and nephrectomized BKO+PBS mice (n = 6). (C) Serum PTH from sham WT (n = 8), nephrectomized WT (n = 9), nephrectomized WT+PBS (n = 7), sham BKO (n = 6), nephrectomized BKO (n = 8), and nephrectomized BKO+PBS mice (n = 5). a p < 0.05 versus sham WT, b p < 0.05 versus nephrectomized WT, c p < 0.05 versus nephrectomized WT+PBS, d p < 0.05 versus sham BKO, and e p < 0.05 versus nephrectomized BKO.

Article Snippet: Serum biochemistries were measured using ELISA kits for erythropoietin (R&D systems, Minneapolis, MN), FGF23 (Quidel, San Diego, CA), and PTH levels (Quidel, San Diego, CA).

Techniques:

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility

doi: 10.1152/ajpendo.00343.2017

Figure Lengend Snippet: Fibroblasti growth factor (Fgf) receptors (Fgfr), α-Klotho, and Fgf23 are expressed in skeletal muscle. A–C: are representative real-time RT-PCR amplification plots showing values of fluorescence at each cycle number of Fgfr1–4, α-Klotho, and Fgf23 (each reaction in triplicate) in isolated extensor digitorum longus (EDL; A) and soleus (B) muscles from 4- to 5-mo-old CD-1 male mice and in differentiated C2C12 myotubes (C). D: summary data showing average 2–ΔCT values (×106) of Fgfr1–4, α-Klotho, and Fgf23 in EDL and soleus muscles from 4- to 5-mo-old CD-1 male mice (n = 3) and in C2C12 myoblasts and myotubes (n = 1) expressed relative to β-actin on log10 scale.

Article Snippet: RNA was also extracted from normal littermate (NL) rat and Cy/+ rat EDL ( n = 6) and C 2 C 12 cells treated with vehicle ( n = 3–6), 100 ng/ml recombinant mouse FGF23 (R & D Systems, Minneapolis, MN; n = 6), or 100 ng/ml recombinant mouse FGF2 (R & D Systems) as a positive control ( n = 3) using the miRNeasy Mini Kit (Qiagen), as previously reported ( 6 ).

Techniques: Quantitative RT-PCR, Amplification, Fluorescence, Isolation, Muscles

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility

doi: 10.1152/ajpendo.00343.2017

Figure Lengend Snippet: FGF23 treatment has no effect on C2C12 proliferation. A and B: cellular proliferation of undifferentiated C2C12 myoblasts (A) and differentiated C2C12 myotubes (B) treated with FGF23 (100 ng/ml), soluble Klotho (1 µg/ml), FGF23 + Klotho, or FGF2 (100 ng/ml, positive control) for 24 and 48 h, as assessed by absorbance at 490 nm using CellTiter 96 AQueous One kit (n = 3; 3 separate experiments). C: MTT assay of undifferentiated C2C12 myoblast proliferation after treatment with FGF23 (2–50 ng/ml) or FGF2 (50 ng/ml) for 24, 48, and 72 h (n = 4; repeated twice). D: hemocytometer-based cell count of undifferentiated C2C12 myoblasts after treatment with FGF23 (2–50 ng/ml) or FGF2 (50 ng/ml) for 24, 48, and 72 h (n = 3; repeated twice). *P < 0.05, **P < 0.01, and ***P < 0.001, 1-way ANOVA with Bonferroni post hoc analysis.

Article Snippet: RNA was also extracted from normal littermate (NL) rat and Cy/+ rat EDL ( n = 6) and C 2 C 12 cells treated with vehicle ( n = 3–6), 100 ng/ml recombinant mouse FGF23 (R & D Systems, Minneapolis, MN; n = 6), or 100 ng/ml recombinant mouse FGF2 (R & D Systems) as a positive control ( n = 3) using the miRNeasy Mini Kit (Qiagen), as previously reported ( 6 ).

Techniques: Positive Control, MTT Assay, Cell Counting

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility

doi: 10.1152/ajpendo.00343.2017

Figure Lengend Snippet: FGF23 treatment does not alter expression of myogenic markers or oxidative stress. A–D: expression of myogenic gene markers Pax7 (A), Myod (B), Myogenin (C), and Myostatin (D) in differentiated C2C12 myotubes after treatment with FGF23 (100 ng/ml) or vehicle for 24 and 48 h. Gene expression was calculated using the 2–ΔΔCT method (n = 6). E: positive control data showing expression of Pax7, Myod, Myogenin, and Myostatin in differentiated C2C12 myotubes after treatment with FGF2 (100 ng/ml) or vehicle for 48 h (n = 3). *P < 0.05 and ***P < 0.001, 1-way ANOVA with Bonferroni post hoc analysis. F: oxidative stress was assessed by measuring the DNA oxidative damage marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) in differentiated C2C12 myotubes after 24-h treatment with FGF23 (100 ng/ml) or vehicle (n = 6). P > 0.05, Student’s t-test.

Article Snippet: RNA was also extracted from normal littermate (NL) rat and Cy/+ rat EDL ( n = 6) and C 2 C 12 cells treated with vehicle ( n = 3–6), 100 ng/ml recombinant mouse FGF23 (R & D Systems, Minneapolis, MN; n = 6), or 100 ng/ml recombinant mouse FGF2 (R & D Systems) as a positive control ( n = 3) using the miRNeasy Mini Kit (Qiagen), as previously reported ( 6 ).

Techniques: Expressing, Positive Control, Marker

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility

doi: 10.1152/ajpendo.00343.2017

Figure Lengend Snippet: Neither acute nor long-term FGF23 treatment has an effect on C2C12 myotube [Ca2+]i. A: representative fluo-4 fluorescent images in C2C12 cells after vehicle, FGF23 (20 ng/ml), KCl (80 mM), or caffeine (20 mM) perfusion. Note the increase in fluorescence after KCl and caffeine treatment but not FGF23. B: representative fluo-4 fluorescence signal and perfusion protocol showing acute C2C12 myotube [Ca2+]i responses to FGF23 (20 ng/ml) (left) and KCl (80 mM) and caffeine (20 mM) perfusion (right). Arrowheads indicate points of perfusion of specific treatments. The fluorescence signal is expressed relative to baseline (F/FO). C: data summary of average peak change in fluo-4 fluorescence after perfusion with FGF23 (20 ng/ml) or vehicle expressed relative to baseline (F/FO) (n = 3 dishes/group with 7–8 myotubes/dish averaged). P > 0.05, Student’s t-test. D: average change in fluo-4 fluorescence in response to perfusion with KCl (80 mM) and caffeine (20 mM) in myotubes incubated with FGF23 (20 ng/ml) for 24 h or 6 days (n = 3–6 dishes/group with 5–11 myotubes/dish averaged). Horizontal dashed line indicates the fluorescence level at baseline before stimulation of Ca2+ release. P > 0.05, 1-way ANOVA. E: Rhod-3 fluorescence in differentiated C2C12 myocytes after treatment with FGF23 (100 ng/ml) for 4 h (n = 6). P > 0.05, Student’s t-test.

Article Snippet: RNA was also extracted from normal littermate (NL) rat and Cy/+ rat EDL ( n = 6) and C 2 C 12 cells treated with vehicle ( n = 3–6), 100 ng/ml recombinant mouse FGF23 (R & D Systems, Minneapolis, MN; n = 6), or 100 ng/ml recombinant mouse FGF2 (R & D Systems) as a positive control ( n = 3) using the miRNeasy Mini Kit (Qiagen), as previously reported ( 6 ).

Techniques: Fluorescence, Incubation

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility

doi: 10.1152/ajpendo.00343.2017

Figure Lengend Snippet: Acute FGF23 administration does not alter CD-1 mouse EDL muscle contractile properties. A: representative ex vivo contractility force data obtained from 1 muscle (x-axis: time; y-axis: force) showing an entire contraction protocol and time of FGF23 addition. B: maximal tetanic force output (at 200-Hz stimulation) from CD-1 mouse EDL muscles after treatment with vehicle or FGF23 expressed relative to values before vehicle or FGF23 application. C: force-frequency relationship of vehicle- or FGF23-treated EDL muscles stimulated to contract with frequencies in the range of 1–220 Hz. Forces at each frequency are expressed relative to the maximal force obtained. D: time course of maximal tetanic force decline during a fatiguing protocol in vehicle- or FGF23-treated EDL muscles. Force at each time point is expressed relative to force just before fatigue. E: maximal tetanic force recovery during various time points postfatigue and with the addition of 5 mM caffeine in vehicle- or FGF23-treated EDL muscles. F: half-maximal tetanic force output (at 100-Hz stimulation) from CD-1 mouse EDL muscles after treatment with vehicle or FGF23 expressed relative to values before vehicle or FGF23 application. G: time course of half-maximal tetanic force decline during a fatiguing protocol in vehicle- or FGF23-treated EDL muscles. Force at each time point is expressed relative to force just before fatigue. H: half-maximal tetanic force recovery during various time points postfatigue and with the addition of 5 mM caffeine in vehicle- or FGF23-treated EDL muscles. I: maximal and half-maximal tetanic force after exposure of EDL muscles to 10 mM caffeine or 5 µM ryanodine, as positive controls, expressed as %force before vehicle or FGF23 treatment (however, error bars that are present in each group may be too small to be seen). Dashed lines in B and F represent points at which FGF23 was added to the muscle contractility bath; n = 3–10 muscles/group. **P < 0.01 and ***P < 0.001, 2-way ANOVA with Bonferroni post hoc analysis.

Article Snippet: RNA was also extracted from normal littermate (NL) rat and Cy/+ rat EDL ( n = 6) and C 2 C 12 cells treated with vehicle ( n = 3–6), 100 ng/ml recombinant mouse FGF23 (R & D Systems, Minneapolis, MN; n = 6), or 100 ng/ml recombinant mouse FGF2 (R & D Systems) as a positive control ( n = 3) using the miRNeasy Mini Kit (Qiagen), as previously reported ( 6 ).

Techniques: Ex Vivo, Muscles

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility

doi: 10.1152/ajpendo.00343.2017

Figure Lengend Snippet: Acute FGF23 administration does not alter CD-1 mouse soleus muscle contractile properties. A: maximal tetanic force output (at 140–160 Hz stimulation) from CD-1 mouse soleus muscles after treatment with vehicle or FGF23 expressed relative to values before vehicle or FGF23 application. B: force-frequency relationship of vehicle- or FGF23-treated soleus muscles stimulated to contract with frequencies in the range of 1–220 Hz. Forces at each frequency are expressed relative to the maximal force obtained. C: time course of maximal tetanic force decline during a fatiguing protocol in vehicle- or FGF23-treated soleus muscles. Force at each time point is expressed relative to force just before fatigue. D: maximal tetanic force recovery during various time points postfatigue and with the addition of 5 mM caffeine in vehicle- or FGF23-treated soleus muscles. E: half-maximal tetanic force output (at 40 Hz stimulation) after treatment with vehicle or FGF23 expressed relative to values before vehicle or FGF23 application. F: time course of half-maximal tetanic force decline during a fatiguing protocol in vehicle- or FGF23-treated soleus muscles. Force at each time point is expressed relative to force just before fatigue. G: half-maximal tetanic force recovery during various time points postfatigue and with the addition of 5 mM caffeine in vehicle- or FGF23-treated soleus muscles. H: maximal and half-maximal tetanic force after exposure of soleus muscles to 10 mM caffeine or 5 µM ryanodine, as positive controls, expressed as a %force before vehicle or FGF23 treatment (however, error bars that are present in each group may be too small to be seen). Dashed lines in A and E represent points at which FGF23 was added to the muscle contractility bath; n = 5–10 muscles/group. ***P < 0.001, 2way ANOVA with Bonferroni post hoc analysis.

Article Snippet: RNA was also extracted from normal littermate (NL) rat and Cy/+ rat EDL ( n = 6) and C 2 C 12 cells treated with vehicle ( n = 3–6), 100 ng/ml recombinant mouse FGF23 (R & D Systems, Minneapolis, MN; n = 6), or 100 ng/ml recombinant mouse FGF2 (R & D Systems) as a positive control ( n = 3) using the miRNeasy Mini Kit (Qiagen), as previously reported ( 6 ).

Techniques: Muscles

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility

doi: 10.1152/ajpendo.00343.2017

Figure Lengend Snippet: Acute FGF23 administration increases isolated heart contractility. A: representative force tracings of isolated mouse heart muscle paced at 1 Hz at baseline (before vehicle or FGF23 treatment) and after 30 min of exposure to vehicle or 9 ng/ml FGF23. B: summary data of whole heart contractile force output after 30 min of vehicle or 9 ng/ml FGF23 exposure. Data are expressed as fold change of contractile force relative to baseline; n = 4–6 hearts/group. *P < 0.05, 2-tailed t-test.

Article Snippet: RNA was also extracted from normal littermate (NL) rat and Cy/+ rat EDL ( n = 6) and C 2 C 12 cells treated with vehicle ( n = 3–6), 100 ng/ml recombinant mouse FGF23 (R & D Systems, Minneapolis, MN; n = 6), or 100 ng/ml recombinant mouse FGF2 (R & D Systems) as a positive control ( n = 3) using the miRNeasy Mini Kit (Qiagen), as previously reported ( 6 ).

Techniques: Isolation